A peer-reviewed open-access journal MycoKeysMultilocus 38: 121–127 (2018)phylogeny reveals taxonomic misidentification of theSchizopora paradoxa... 121 doi: 10.3897/mycokeys.38.28497 SHORT COMMUNICATION MycoKeys http://mycokeys.pensoft.net Launched to accelerate biodiversity research

Multilocus phylogeny reveals taxonomic misidentification of the Schizopora paradoxa (KUC8140) representative genome

Javier Fernández-López1, María P. Martín1, Margarita Dueñas1, M. Teresa Telleria1

1 Departmento de Micología, Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Corresponding author: Javier Fernández-López ([email protected])

Academic editor: T. Lumbsch | Received 20 July 2018 | Accepted 13 August 2018 | Published 28 August 2018

Citation: Fernández-López J, Martín MP, Dueñas M, Telleria MT (2018) Multilocus phylogeny reveals taxonomic misidentification of theSchizopora paradoxa (KUC8140) representative genome. MycoKeys 38: 121–127. https://doi. org/10.3897/mycokeys.38.28497

Abstract Schizopora paradoxa, current name Xylodon paradoxus, is a white-rot with certain useful biotechno- logical properties. The representative genome of Schizopora paradoxa strain KUC8140 was published in 2015 as part of the 1000 Fungal Genomes Project. Multilocus phylogenetic analyses, based on three nu- clear regions (ITS, LSU and rpb2), confirmed a misidentification of S. paradoxa strain KUC8140 which should be identified asXylodon ovisporus. This wrong identification explains the unexpected geographical distribution of S. paradoxa, since this species has a European distribution, whereas the strain KUC8140 was recorded from Korea, Eastern Asia.

Keywords , phylogenetic analyses, , white-rot fungi, Xylodon

Introduction

The Schizopora Velen., currently synonymous with Xylodon (Pers.) Fr. (Riebesehl and Langer 2017), includes white-rot fungi that play an important role in ecosys- tem processes as a wood decomposer. The description and identification of Xylodon (=Schizopora) species, based on morphological characters, has led to inaccuracies due to a lack of clear diagnostic characters and it has been assumed that many Xylodon species have a worldwide distribution (Paulus et al. 2000). However, during the last

Copyright Javier Fernández-López et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 122 Javier Fernández-López et al. / MycoKeys 38: 121–127 (2018) decade, it has been pointed out that fungal cosmopolitanism could be the result of the application of a morphological species recognition criterion and not the result of an actual biogeographical pattern (Taylor et al. 2006). Moreover, phylogenetic analyses have revealed an undescribed species diversity masked by the morphological species recognition approach (Taylor et al. 2000). The representative genome of Schizopora paradoxa strain KUC8140, current name Xylodon paradoxus (Schrad.) Chevall., was sequenced in 2015 as part of the 1000 Fun- gal Genomes Project (http://jgi.doe.gov/fungi) (Min et al. 2015); this strain was col- lected from an oak forest in Korea. Usually X. paradoxus has been associated with late stages of wood decay, mainly in deciduous forests and shows useful biotechnological properties for bioremediation, such as tolerance to heavy metals or dye decolourising activity (Lee et al. 2014). It has been recorded around the world; however, available ge- netic data point to a European distribution (Paulus et al. 2000). Within the framework of a broader study of Xylodon through molecular approaches, the taxonomic identity of the strain KUC8140 has been assessed.

Materials and methods

In order to infer the taxonomic position of the strain KUC8140, phylogenetic rela- tionships of six Xylodon species were addressed. DNA from specimens of X. paradoxus, X. quercinus (Pers.) Gray, X. nothofagi (G. Cunn.) Hjorstam & Ryvarden, X. raduloides Riebesehl & E. Langer, X. flaviporus (Ber. & M.A. Curtis ex Cooke) Riebesehl & E. Langer and X. ovisporus (Corner) Riebesehl & E. Langer was extracted from herbaria specimens and culture collections (Table 1). Three specimens of the sister genusLyo - myces P. Karst. were included as outgroup in the phylogenetic analyses (Table 1). DNA isolation was performed using DNeasy™ Plant Mini Kit (Qiagen, Valencia, California, USA) following the manufacturer’s instructions. Three nuclear regions were amplified and sequenced: nuclear ribosomal internal transcribed spacer (ITS, fungal barcoding; Schoch et al. 2012), nuclear large ribosomal subunit (LSU) and the second largest subunit of RNA polymerase II (rpb2). Direct Polymerase chain reactions (PCRs) were performed to obtain sequences from ITS and LSU with the pair of primers ITS5/ITS4 (White et al. 1990) and LR0R/LR5 (Rehner and Samuels 1994), respectively. Nested- PCRs were done to obtain amplifications of rpb2 fragments, using RPB2-5F/RPB2- 7.1R (Liu et al. 1999, Matheny 2005) for the first amplification followed by RPB2-6F/ RPB2-7R2 (Matheny et al. 2007), using 1 μl of the first PCR as target DNA. Ampli- fications were undertaken using illustra™ PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare, Buckinghamshire, UK) as described in Winka et al. (1998), following thermal cycling conditions in Martín and Winka (2000). Negative controls lacking fungal DNA were run for each experiment to check for contamination. Amplifications were assayed by gel electrophoresis in 2% Pronadisa D-1 Agarose (Lab. Conda, Tor- rejón de Ardoz, Spain). Amplified DNA fragments were purified from the agarose gel using the Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madi- son, WI, USA) and sent to Macrogen Korea (Seoul, Korea) for sequencing. Primers, Multilocus phylogeny reveals taxonomic misidentification of theSchizopora paradoxa... 123

Table 1. Specimen information, GenBank accession numbers and genome BLAST searches (ID) used in this study. New sequences generated in this study are indicated in bold. n.d.: no data.

GenBank accession number Species Specimen voucher Country ITS LSU rpb2 HHB 10401 USA MH260068 MH260061 MH259316 Lyomyces crustosus HHB 13100 USA MH260069 MH260062 MH259317 UC 2022841 USA KP814310 n.d. n.d. ICMP 13836 Taiwan AF145585 n.d. n.d. Xylodon flaviporus MA-Fungi 79440, 12094IS Germany MH260071 MH260066 MH259319 ICMP 13839 New Zealand AF145582 MH260064 MH259322 Xylodon nothofagi PDD 91630, BCP 3306 New Zealand GQ411524 n.d. n.d. ICMP 13835 Taiwan AF145586 MH260063 MH259320 Xylodon ovisporus ICMP 13837 Taiwan AF145587 n.d. n.d. FCUG 2425 Russia AF145577 n.d. n.d. Xylodon paradoxus MA-Fungi 70444, 11060MD France MH260070 MH260065 n.d. MA-Fungi 81294, 13833MD France MH260072 n.d. MH259318 H 6013352 Finland KT361632 n.d. n.d. Xylodon quercinus MA-Fungi 91311, 1JFL Spain MH260073 MH260067 MH259321 ICMP 13833 Australia AF145580 KY962853 n.d. Xylodon raduloides MA-Fungi 75310, GP2291 Spain KY962825 KY962864 KY967055 Schizopora paradoxa KUC8140 Korea ID14957398 ID14957349 ID1495735 used for sequencing, were those used for PCR amplifications. Additional searches for the six Xylodon species in EMBL/GenBank/DDBJ databases were performed in order to complete the molecular information available for this genus. Using the BLAST tool from the JGI portal, ITS, LSU and rpb2 sequences were extracted from the KUC8140 strain genome (https://genome.jgi.doe.gov/pages/blast- query.jsf?db=Schpa1). The same regions from X. paradoxus specimens FCUG-2425, MA-Fungi 70444 and MA-Fungi 81294 were used as reference sequences for BLAST searches, respectively (Table 1). For ITS and LSU, custom search settings were used (blastn; all databases; Expect = 1*10-3; Word size = 11; Filter low complexity regions; Scoring matrix = PAM30; ITS Job ID = 14957398; LSU Job ID = 14957349). For rpb2, default BLAST settings were used (blastn; assembly database; Expect = 1*10-5; Word size = 11; Filter low complexity regions; Scoring matrix = BLOSUM62; rpb2 Job ID = 14957357). The best scoring sequence from theS. paradoxa KUC8140 strain genome for each region was extracted and downloaded. Raw sequence data were processed and assembled with Geneious version 9.0.2. (Kearse et al. 2012). Two individual datasets, ITS-LSU concatenated and rpb2, were created to compare the KUC8140 strain with other Xylodon species. The combination of novel, GenBank and KUC8140 sequences for each dataset were aligned in Geneious 9.0.2 with the MAFFT nucleotide sequence alignment function (Katoh and Standley 2013). The automatic alignments were reviewed manually through Geneious 9.0.2. Phylogenetic tree estimation for each alignment was performed using Maximum Likelihood (ML) and Bayesian Inference (BI). ML and bootstrapping analyses were conducted in RAxML (Stamatakis 2006), using default parameters established in the 124 Javier Fernández-López et al. / MycoKeys 38: 121–127 (2018)

CIPRES web portal (http://www.phylo.org/portal2/; Miller et al. 2010) and calculat- ing bootstrap statistics from 1000 replicates. Bayesian inference analyses were imple- mented in BEAST v2.4.3 (Drummond and Rambaut 2007). Site model partition was selected using jModelTest2 (Darriba et al. 2012) and defined using BEAUti v2.4.3 interface. HKY and GTR substitution models were selected for ITS+LSU and rpb2 alignments, respectively, as the closest available in BEAST from the results obtained in jModelTest2. We used relative timing with an uncorrelated lognormal relaxed clock by calibrating the tree with a value of 1 in the root for the Xylodon clade. Birth Death model was used as a tree prior. One MCMC run was specified for 50 million gen- erations, sampling every 5000th generation. Results were visualised in Tracer v.1.6 (Rambaut et al. 2018) to evaluate whether the effective sample size (ESS) values were above 200. The trees obtained were summarised in a maximum clade credibility tree by TreeAnnotator v.1.7. with a burn-in of 5000.

Results and discussion

The ITS+LSU dataset was 1193 characters long (ITS = 594; LSU = 599) and therpb 2 dataset was 647 characters long. The results of phylogenetic analyses of ITS+LSU and rpb2 datasets are summarised in Fig. 1, using phytools R package (Revell 2012). Each phylogram represents the best tree produced from the RAxML analysis. All effective sample sizes from BEAST analyses were higher than 200 for all parameters. Those clades with Maximum likelihood bootstrap (MLB) percentages ≥ 75% and Bayesian posterior probabilities (BPP) ≥ 0.99 are marked with empty circles in Fig. 1. The re- maining support values are represented above branches (MLB/BPP); specimen vouch- ers and species names are provided on the tip labels. Our phylogenetic analyses confirmed the misidentification of S. paradoxa strain KUC8140, since sequences of this strain grouped in the X. ovisporus clade, showing a dif- ferent evolutionary history from X. paradoxus. Therefore, S. paradoxa strain KUC8140, from Korea, must be identified asXylodon ovisporus, reported from Asia and West Pacific areas (Wu 2000, Hattori 2003). The new identity of the strain KUC8140 is also sup- ported by geographical data, since S. paradoxa has a European distribution. This rectifi- cation helps to explain the biogeographical patterns of Xylodon and also sustains the idea that “not everything is everywhere” for wood-decay fungi (Lumbsch et al. 2008). According to our phylogenetic analyses, Xylodon ovisporus is the sister species of X. flaviporus and morphological characters confirm this relationship. The species can be discriminated by the spore size, shorter in the first one (Hattori 2003). This example accords with studies that warn about misidentifications or mislabelled vouchers in public sequence databases (Bidartondo 2008). It has been estimated that around 20% of DNA fungal sequences in the GenBank repository may have erroneous lineage as- signations (Bridge et al. 2003, Nilsson et al. 2006). Assessing accuracy in GenBank and other DNA repositories is a key stage for species identification in current biodiversity analyses based on similarity of DNA sequences (Hibbett et al. 2016). It is especially Multilocus phylogeny reveals taxonomic misidentification of theSchizopora paradoxa... 125

Figure 1. Maximum likelihood trees for ITS+LSU (left) and rpb2 (right) regions of Xylodon species. In order to assess genealogical concordance, dotted lines link the position of the same specimen in both trees. Grey boxes indicate the position of KUC8140 strain with Xylodon ovisporus and the position of X. paradoxus. Numbers over branches are maximum likelihood bootstrap (MLB) values and posterior prob- abilities (BPP). Voucher numbers and species names are indicated in Table 1. important in cases like Xylodon paradoxus, with useful biotechnological properties since, according to Bortolus (2008), a wrong taxonomy could lead not only to inac- curate knowledge of nature, but also to important economic losses.

Acknowledgments

This work was supported by the Plan Nacional I+D+i projects n° CGL2012-35559, CGL2015-67459-P. J.F.L. was supported by a Predoctoral Grant from the Ministerio de Economía y Competitividad (Spain) (BES-2013-066429). Karl-Henrik Larsson provided com- ments and suggestions that improved this manuscript. Thanks to M. Glenn (Seton Hall University, US) for the English revision. Also to the staff of ICMP, PDD and the Madison Forest Products Laboratory (USDA) for their invaluable assistance.

References

Bidartondo MI (2008) Preserving accuracy in GenBank. Science 319: 1616. https://doi. org/10.1126/science.319.5870.1616a 126 Javier Fernández-López et al. / MycoKeys 38: 121–127 (2018)

Bortolus A (2008) Error cascades in the Biological Sciences: The unwanted consequences of using bad taxonomy in ecology. AMBIO: A Journal of the Human Environment 37: 114– 118. https://doi.org/10.3410/f.1112162.568148 Bridge PD, Roberts PJ, Spooner BM, Panchal G (2003) On the unreliability of pub- lished DNA sequences. The New Phytologist 160: 43–48. https://doi.org/10.1046 /j.1469-8137.2003.00861 Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772–772. https://doi.org/10.1038/nmeth.2109 Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. https://doi.org/10.1186/1471-2148-7-214 Hattori T (2003) Type studies of the polypores described by E.J.H. Corner from Asia and West Pacific Areas. V. Species described inTyromyces (2). Mycoscience 44: 265–276. https://doi. org/10.1007/s10267-003-0114-3 Hibbett D, Abarenkov K, Kõljalg U, Öpik M, Chai B, Cole J et al. (2016) Sequence-based classification and identification of Fungi. Mycologia 108: 1049–1068.https://doi. org/10.3852/16-130 Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Im- provements in Performance and Usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010 Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al. (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649. https://doi.org/10.1093/bioin- formatics/bts199 Lee H, Jang Y, Choi Y-S, Kim M-J, Lee J, Lee H et al. (2014) Biotechnological procedures to select white rot fungi for the degradation of PAHs. Journal of Microbiological Methods 97: 56–62. https://doi.org/10.1016/j.mimet.2013.12.007 Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Lumbsch HT, Buchanan PK, May TW, Mueller GM (2008) Phylogeography and biogeog- raphy of Fungi. Mycological Research 112: 423–424. https://doi.org/10.1016/j.my- cres.2008.02.002 Martín MP, Winka K (2000) Alternative methods of extracting and amplifying DNA from lichens. The Lichenologist 32: 189–196.https://doi.org/10.1006/lich.1999.0254 Matheny PB (2005) Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe; Agaricales). Molecular Phylogenetics and Evolution 35: 1–20. https://doi.org/10.1016/j.ympev.2004.11.014 Matheny PB, Wang Z, Binder M, Curtis JM, Lim YW, Nilsson RH et al. (2007) Contri- butions of rpb2 and tef1 to the phylogeny of mushrooms and allies (, Fungi). Molecular Phylogenetics and Evolution 43: 430–451. https://doi.org/10.1016/j. ympev.2006.08.024 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE). IEEE, New Orleans, LA, 1–8. https://doi.org/10.1109/GCE.2010.5676129 Multilocus phylogeny reveals taxonomic misidentification of theSchizopora paradoxa... 127

Min B, Park H, Jang Y, Kim J-J, Kim KH, Pangilinan J et al. (2015) Genome sequence of a white rot fungus Schizopora paradoxa KUC8140 for wood decay and mycoremediation. Journal of Biotechnology 211: 42–43. https://doi.org/10.1016/j.jbiotec.2015.06.426 Nilsson RH, Ryberg M, Kristiansson E, Abarenkov K, Larsson K-H, Koljalg U (2006) Taxo- nomic reliability of DNA sequences in public sequence databases: A fungal perspective. PLoS ONE 1(1): e59. https://doi.org/10.1371/journal.pone.0000059. Paulus B, Hallenberg N, Buchanan PK, Chambers GK (2000) A phylogenetic study of the ge- nus Schizopora (Basidiomycota) based on ITS DNA sequences. Mycological Research 104: 1155–1163. https://doi.org/10.1017/S0953756200002720 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Tracer v1.7. https://doi. org/10.1093/sysbio/syy032 Rehner SA, Samuels GJ (1994) Taxonomy and phylogeny of Gliocladium analysed from nu- clear large subunit ribosomal DNA sequences. Mycological Research 98: 625–634. https:// doi.org/10.1016/S0953-7562(09)80409-7 Revell LJ (2012) phytools: an R package for phylogenetic comparative biology (and other things). Methods in Ecology and Evolution 3: 217–223. https://doi.org/10.1111/j.2041- 210X.2011.00169.x Riebesehl J, Langer E (2017) Hyphodontia s.l. (Hymenochaetales, Basidiomycota): 35 new combinations and new keys to all 120 current species. Mycological Progress 16: 637–666. https://doi.org/10.1007/s11557-017-1299-8 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 6241–6246. https://doi.org/10.4172/2169-0111.1000119 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690. https://doi. org/10.1093/bioinformatics/btl446 Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS et al. (2000) Phyloge- netic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21–32. https://doi.org/10.1006/fgbi.2000.1228 Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D (2006) Eukaryotic microbes, spe- cies recognition and the geographic limits of species: examples from the kingdom Fungi. Philosophical Transactions of the Royal Society of London 361: 1947–63. https://doi. org/10.1098/rstb.2006.1923 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ri- bosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White YJ (Eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Winka K, Ahlberg C, Eriksson OE (1998) Are there Lichenized Ostropales? The Lichenologist 30: 455–462. https://doi.org/10.1006/lich.1998.0142 Wu SH (2000) Studies on Schizopora flavipora s.l., with special emphasis on specimens from Taiwan. Mycotaxon 76: 51–66. https://doi.org/10.2307/3761766